The field of the invention is detection and quantification of oxidative stress in a subject.
It is by now common knowledge that stress in mammalian subjects develops directly or indirectly into a display of oxygenated species, which tends to change the usually reduced state of the body to a hyperoxygenated state. This hyperoxygenated state includes generation and reaction of hydroxides, peroxides and free radical species, which are thought to be implicated in physiological imbalance and actual physical damage. Physical damage can produce pathological states, which for example, may lead to atherosclerotic plaques. Such plaques often result in the deposition of lipids and may further lead to blockage of arteries that can cause a cessation of blood flow to the heart with a resulting heart attack. This is one of many human disease states that are thought to be caused by free radical attack from the hyperoxygenated state caused by stress. Despite the relatively large body of information linking oxidative stress to various diseases and/or disease states, there is still an appreciable need for suitable markers and test systems to determine the level of oxidative stress in a simple and inexpensive manner.
Malondialdehyde is a component of normal urine, and its presence can be determined using relatively expensive and typically stationary equipment such as spectrophotometers, fluorometers, high performance liquid chromatographs and gas chromatograph mass spectrometers. Such equipment typically enables an operator to determine not only the quantity of a particular aldehyde, but also to determine the chemical nature of a particular molecule with an aldehyde function. Unfortunately, the operator of such equipment needs to be highly trained, and the weight and size of the equipment is generally prohibitive for point-of-care tests.
Alternatively, a broad spectrum of chemically distinct aldehydes may be detected by mixing a drop of sample solution (which may contain the aldehyde) with 2 ml of 72 percent sulfuric acid in a test tube (disclosed at page 395 in xe2x80x9cQualitative Analysis by Spot Testsxe2x80x9d, Third Edition, authored by F. Feigl and published by Elsevier Publishing Company, Inc.). A small amount of solid chromatropic acid (1,8-dihydroxynapthlanene-3,6-disulfate) is added to the mixture, and the test tube is heated in a 60xc2x0 C. water bath for about ten minutes. If an aldehyde is present, a bright violet color appears in the test solution. While the test is relatively non-specific for a particular aldehyde, the sensitivity of the test is reportedly about 3 ppm. However, the reaction mixture typically requires vigorous heating for at least 10 minutes to provide an at least semi-quantitative and reliable test result.
In yet another method generally applicable to aldehydes, described at pages 339-340 of the Feigl publication, a drop of aqueous (or alcoholic) solution suspected of containing an aldehyde is treated on a spot plate with a drop of sulfurous acid and a drop of fuchsin/sulfuric acid and allowed to react on the plate. A red to blue color appears within about two to thirty minutes, according to the amount of aldehyde present in the test solution being tested. Such test is reportedly sensitive to about one microgram of formaldehyde in the drop of solution being tested. Although the fuchsin/sulfuric acid reaction can advantageously be performed at room temperature, the test results tend to vary depending on the time allowed for the reaction.
Although various quantitative and qualitative tests for aldehydes are known in the art, all or almost all of them suffer from one or more disadvantage. Moreover, despite the existence of known tests, it has never been appreciated that such tests can be applied to malondialdehyde in urine to detect oxidative stress. Thus, there is still a need to provide methods and apparatus for detecting oxidative stress in subjects.
It has been discovered that the oxidative stress state of a person can be measured from the release into the urine of an aldehyde, and particularly malondialdehyde, and that an aldehyde-reactive chromogen based calorimetric test can measure the released aldehyde in a rapid, easily performed test.
In particular, a method of determining oxidative stress in a subject has one step in which presence of an aldehyde in a biological fluid of a subject is correlated with an oxidative stress in the subject. In another step, a test reagent comprising a pH regulator, a reducing agent, and an aldehyde-reactive chromogen is provided, and the test reagent is combined with the biological fluid to produce an aldehyde-modified chromogen. In yet another step, a color of the aldehyde-modified chromogen is correlated with the oxidative stress.
In one aspect of the inventive subject matter, any biological fluid is considered suitable for use with the test, and especially preferred fluids include saliva, serum, plasma, and spinal fluid, most preferably urine. It is further contemplated that such fluids are derived from a mammalian system (e.g., human, live stock, pet, or cell culture).
In a further aspect of the inventive subject matter, the aldehyde comprises a dialdehyde, and especially contemplated dialdehydes include malondialdehyde. Particularly preferred pH regulators comprise a buffer or an acid, such as phosphoric acid and/or glacial acetic acid, and reducing agents typically have a sulfur (e.g., sodium metabisulfide) and/or phosphorous atom (e.g., TCEP). Further preferred aldehyde-reactive chromogens (e.g., fuchsin) include a reactive group that selectively reacts with an aldehyde and thereby shift their absorption maximum towards higher or lower wavelength in a concentration dependent manner.